The cost of DNA sequencing is still generally too expensive for routine applications. For example, the estimated cost, including conventional instrumentation, sample preparation and labor, for sequencing a haploid human genome ranges from $100,000 to $1,000,000. Despite the costs involved, it is expected that the demand for sequence information will keep increasing.
One proposed solution has been to perform DNA sequencing using biomolecule or DNA nanoparticle translocation through a nanopore. Nanoparticle translocation is an active, robust, repeatable and controllable engineering approach that has both fundamental and practical significances in a large number of scientific fields including genetics, biochemistry, biophysics, chemistry, physiochemistry, biomedical science, clinic diagnostics, molecular biology, evolutionary biology, and anthropology.
With respect to DNA sequencing, nanopore translocation would be used as follows. First, a translocation device is provided, including first and second reservoirs and a nanopore providing fluid communication between the reservoirs. The first and second reservoirs are configured to include cathode and anode electrodes, respectively. Further, the reservoirs and nanopore are filled with an aqueous electrolyte. Second, the DNA of interest is introduced into the first reservoir. Third, a DC voltage is imposed across a nanopore submerged in an aqueous electrolyte, resulting in an ionic current through the nanopore and electrophoresis of the DNA through the nanopore. Finally, the ionic current during translocation is measured to ascertain the sequence for the DNA in the first reservoir.
In general, the current through such a nanopore is very sensitive to the size and shape of the nanopore. Therefore, if single bases or strands of DNA pass (or part of the DNA molecule passes) through the nanopore by electrophoresis, this can create a measurable change in the magnitude of the ionic current through the nanopore. This ionic current through the nanopore can be measured using conventional electrophysiological techniques. The ionic current is also affected by a DNA obstructing the nanopore and returns to a baseline current after the DNA exits the nanopore. In particular, since the A, C, G, and T nucleotides on a DNA molecule carry different surface charges, each nucleotide may alter the ionic current through the nanopore to a different characteristic degree. Accordingly, the amount of current which can pass through the nanopore at any given moment can vary depending on whether the nanopore is blocked by an A, a C, a G or a T nucleotide. Therefore, the change in the current through the nanopore as the DNA molecule passes through the nanopore represents a direct reading of the DNA sequence. As a result, it has been hypothesized that the sequence of bases in DNA can be recorded by monitoring such current modulations.
Although nanopore translocation-based DNA sequencing technologies appear to provide a solution for reducing costs of DNA sequencing, a practical system is still unavailable. A primary issue has been the lack of an ability to regulate the translocation process to achieve a nanometer-scale spatial accuracy. That is, the current resolution of ionic current detection systems is too low for the DNA translocation velocities. Accordingly, many efforts have focused on determining how to slow down DNA translocation through the nanopore by modifying viscosity, temperature, and voltage bias. However, such methods can result in a decrease of the signal to noise ratio, making detection of ionic currents difficult, or in a reduction of DNA attraction to the nanopore, thus lowering the overall throughput. Other efforts have focused on adjustment of equipment and/or the electrolyte, but methods typically require additional equipment or additional investigation.